Novel antibiotic NK84-0218 and process for the production of the same

ABSTRACT

This invention relates to a novel antibiotic NK84-0218 of the formula: ##STR1## which exhibits antibacterial, antiviral and antineo-plastic activities and is expected as a pharmaceutical as well as a process for the production of the same.

This application is a division of application Ser. No. 796,114, filedNov. 8, 1985, now U.S. Pat. No. 4,743,689.

BACKGROUND OF THE INVENTION

Antineoplastic adenine antibiotics such as cordycepin(3'-deoxyadenosine;cf. Bio-chim. Biophys. Acta, 117, 482 (1966)) have been known. Up tonow, no compound wherein an oxetane ring is attached to adenine has beenreported.

By the way, with the appearance of resistant bacteria it is alwaysrequired to develop novel antibiotics. Further there are various kindsof viral diseases and malignant tumors, which also makes novel antiviraland antineoplastic active substances necessary.

SUMMARY OF THE INVENTION

Under these circumstances, we have examined various metabolites ofmicroorganisms and consequently found that a strain belonging to thegenus Bacillus produces a novel antibiotic NK84-0218 of the formula (I):##STR2## which exhibits antibacterial, antiviral and antineoplasticactivities.

The present invention has been completed based on the above finding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ultraviolet absorption spectra of NK84-0218. FIG. 2 showsan infrared absorption spectrum of NK84-0218 in the form of potassiumbromide tablets. FIG. 3 shows a ¹ H-nuclear magnetic resonance spectrumof NK84-0218 determined with an apparatus at 400 MHz with the use oftetramethylsilane in deuterated dimethyl sulfoxide as an internalreference.

DETAILED DESCRIPTION OF THE INVENTION

The novel antibiotic NK84-0218 described above can be obtained byculturing an NK84-0218-producing bacterium belonging to the genusBacillus to thereby allow the microorganism to accumulate the antibioticNK84-0218 and collecting the antibiotic NK84-0218 from the medium. Atypical example of the NK84-0218-producing bacterium is BacillusNK84-0218 (FERM BP-919 deposited at the Fermentation Research InstituteAgency of Industrial Science and Technology, 1-3, Higashi 1-chomeYatabe-machi Tsukuba-gun Ibaraki-ken 305, Japan; hereinafter referred toas NK-84-0218 strain) which was isolated from soil in the DirectorResearch Laboratories of Nippon Kayaku Co., Ltd. in December, 1983.

Now mycological properties of the NK84-0218 strain will be described.

1. Morphological properties

(1) Cell form: rod.

Cell size: 1.0-1.3×1.5-4.7μ.

(2) Polymorphism: Observed. This bacterium is cylindrical at an earlystage of the cultivation and then turns to elliptic like an egg with theprogress of the cultivation.

(3) Motility: None.

(4) Spores: Elliptic spores of 0.6-0.8×1.2-1.5μ in size are observed atthe middle, subterminal or terminal site.

(5) Gram staining: Positive.

(6) Acid-fastness: None.

2. Growth in various media (cultured in each medium at 27° C. for one toseven days and observed in a conventional manner)

(1) Bouillon agar plate culture

The bacterium shows an excellent growth. It is in the form of a circleat first and then turns into an irregularly framed shape withmultiplication. A colony thereof is glossy and milk white to paleyellow. No soluble pigment is observed.

(2) Bouillon agar slant culture

The growth surface is smooth at first and turns to wrinkled as thecultivation proceeds. It is opaque and adhesive. No pigment is formed.

(3) Bouillon liquid culture

No growth is observed on the surface. A slight turbidity is observed anda precipitate is observed at the bottom of the tube as the cultivationproceeds. No gas is evolved.

(4) Bouillon gelatin stab culture

Liquid layers are formed with the growth of the bacterium. Noprecipitate is observed.

(5) Litmus milk culture

The medium coagulates at 37° C. Peptonization is observed as thecultivation proceeds and the litmus turns to pale red.

3. Physiological properties

(1) Reduction of nitrate: Negative.

(2) Denitrification: Postive.

(3) MR test: Quasi-positive.

(4) VP test: negative.

(5) Indole formation: Negative.

(6) Hydrogen sulfide formation: Negative.

(7) Hydrolysis of starch: Positive.

(8) Utilization of citric acid: Positive.

(9) Utilization of inorganic nitrogen sources:

It does not seem to utilize inorganic nitrogen sources.

(10) Formation of pigments

King A, B: Both negative.

(11) Urease: Negative.

(12) Oxidase: Positive.

(13) Catalase: Positive.

(14) Growth range

Temperature: 10° to 45° C. (cultured at various temperatures for 30days).

pH: 5 to 10.

(15) Attitude for oxygen: Aerobic.

(16) O-F test: Oxidation.

(17) Formation of acids and gases from various sugars:

Table 1 shows whether this bacterium produces acids and/or gases fromeach sugar.

                  TABLE 1                                                         ______________________________________                                        No.   Sugars     Formation of acids                                                                          Formation of gases                             ______________________________________                                        1     L-arabinose                                                                              +             -                                              2     D-xylose   +             -                                              3     D-glucose  +             -                                              4     D-mannose  -             -                                              5     D-fructose +             -                                              6     D-galactose                                                                              +             -                                              7     marutose   +             -                                              8     sucrose    +             -                                              9     lactose    +             -                                              10    trehalose  +             -                                              11    D-solbitol -             -                                              12    D-mannitol +             -                                              13    inositol   -             -                                              14    glycerin   +             -                                              15    starch     +             -                                              ______________________________________                                         (Used medium is a medium containing 0.5% agar in liquid medium.)         

Based on these findings, this bacterium has been identified as a strainbelonging to Bacillus megaterium by reference to Bergey's Manual ofDeterminative Bacteriology (the 8th ed.) and named Bacillus magateriumNK84-0218.

Similar to other strains belonging to the genus Bacillus, the Bacillusmegaterium strain used in the present invention is labile in properties.Thus mutants thereof can be readily prepared by various artificialmethods such as exposure to ultraviolet light or X-ray, or usingchemical agents. Any mutants capable of producing the antibioticNK84-0218, which is the object of the present invention, can be used inthe present invention.

In order to produce the antibiotic NK84-0218 according to the presentinvention, the abovementioned strain is aerobically cultured in a mediumcontaining nutrients which the microorganism can utilize. Well-knownnutritional sources conventionally used in culturing bacteria can beemployed. That is, carbon sources such as glucose, lactose, glycerol,sucrose, dextrin, galactose, organic acids and combinations thereof; andinorganic and organic nitrogen sources such as ammonium chloride,ammonium sulfate, urea, ammonium nitrate, sodium nitrate, peptone, meatextract, yeast extract, dry yeast, corn steep liquor, soybean powder,cotton seed meal, casamino acids, Bacto-Soytone (an enzymatic hydrolysisof soybean meal, mfd. by Difco Co., Ltd.), soluble vegetable protein,oatmeal and combinations thereof may be employed. Furthermore inorganicsalts such as common salt, calcium carbonate, magnesium sulfate, coppersulfate, ferrous sulfate, zinc sulfate, manganese chloride andphosphates as well as organic compounds such as amino acids, vitaminsand nucleic acids and inorganic compounds may be added if required.

Liquid culture, in particular submerged agitation culture, is the mostsuitable. It is preferable to culture the strain at 20° to 45° C. undera slightly acidic to slightly alkaline condition. This strain usuallyaccumulates the aimed NK84-0218 in a liquid medium when cultured thereinfor one to four days. When the amount of the product reaches themaximum, the culture is ceased and the culturing broth obtained byfiltering off the cells is purified to thereby isolate the aimedsubstance.

The amount of NK84-0218 accumulated in the culturing broth is determinedby high performance liquid chromatography wherein a Nucleosil ₅ C₁₈column (4.6φ×250 mm) and a solvent (0.1M citrate buffer solution (pH4.0)/acetonitrile/methanol=100:4:2) are employed at a flow rate of 0.8ml/min at 21° C. and at 259 nm.

In order to isolate the antibiotic of the present invention, theculturing broth is purified in a manner conventionally used in isolatinga metabolite of a microorganism from its culturing broth. NK84-0218 issoluble in water, methanol and dimethyl sulfoxide and hardly or notsoluble in other conventional organic solvents such as propanol andacetone, so that it is purified by a process conventionally employed topurify nucleoside antibiotics, i.e. adsorption/desorption with activecarbon and ion exchange resins, column chromatography with the use ofAvicel® or Sephadex® or a combination of these procedures. For example,the culturing broth is adjusted to a pH value of 3.5 to 6.5, preferably4 to 4.5, adsorbed by powdery active carbon, washed with water andeluted with a 50% aqueous acetone and the active fraction thus obtainedis concentrated and lyophilized to give a brown crude powder. Then thispowder is dissolved in methanol and insoluble substances are removed.The soluble portion is dried, dissolved in water, adsorbed by an activecarbon column, washed with water and subjected to acetone linearconcentration gradient elution with water and 50% aqueous acetone. Theobtained active fraction is concentrated and lyophilized. The pale brownpowder containing NK84-0218 thus obtained is dissolved in a small amountof water and packed into an Avicel column. Then it is developed with anaqueous alcohol and eluted with increasing the water content stepwise.The active fractions thus obtained are combined, concentrated andlyophilized. Subsequently the obtained powder is dissolved in a smallamount of water and packed into an SP Sephadex® C-25(Na⁺) columnpreviously equilibrated with 0.02M sodium chloride. The active fractionseluted with 0.02M sodium chloride are desalted on active carbon to givea colorless powder. The powder thus purified is treated with water or anaqueous alcohol to give NK84-0218 in the form of colorless needles. Thetiter during the cultivation is determined by plate cup assay withBacillus subtilis PCI 219.

Alternately the substance of the present invention may be purified andisolated in the following manner. That is, the culturing filtrate isadjusted to a pH value of 4 to 4.5, adsorbed by a strongly acidic cationexchange resin, washed with water and eluted with diluted aqueousammonia to give active fractions containing NK84-0218 which exhibits anantibacterial activity. These active fractions are combined, neutralizedand passed through porous resin column. After washing with water andeluting with aqueous methanol, the obtained active fractions areconcentrated in vacuo to give a crude powder containing NK84-0218. Theobtained crude powder is dissolved in methanol and methanol is distilledoff in vacuo from the soluble portion. The residue is passed through aweakly acidic cation exchange resin, washed with water and eluted withdiluted aqueous ammonia. The obtained active fractions are combined andconcentrated in vacuo to thereby remove the ammonia. Then it is allowedto stand in an icebox overnight to give crude crystals of NK84-0218.After recrystallizing from water, NK84-0218 is obtained in the form ofcolorless needles.

The NK84-0218 thus obtained has the following physicochemicalproperties.

(1) Appearance Colorless needles.

(2) Elemental analysis (%) (as C₁₀ H₁₃ O₃ N₅. H₂ O)

    ______________________________________                                                   C    H          O      N                                           ______________________________________                                        calculated:  44.60  5.62       23.77                                                                              26.01                                     found:       44.75  5.58       23.65                                                                              25.98                                     ______________________________________                                    

(3) Molecular formula (molecular weight) C₁₀ H₁₃ O₃ N₅ (251.24)

(4) Melting point 197° C.

(5) Specific rotation [α]=-44.3° (C 0.21, pyridine)

(6) Ultraviolet absorption spectrum

FIG. 1 shows ultraviolet absorption spectra of this substance.

Ultraviolet absorbances and molecular extinction coefficients in water,0.1N hydrochloric acid and 0.1N sodium hydroxide are as follows,respectively:

    ______________________________________                                        λ .sub.max .sup.H.sbsp.2.sup.O (logε)                                          =          259 nm (4.10)                                      λ .sub.max .sup.0.1N HCl (logε)                                                =          257 nm (4.09)                                      λ .sub.max .sup.0.1N NaOH (logε)                                               =          259 nm (4.11).                                     ______________________________________                                    

(7) Infrared absorption spectrum

FIG. 2 shows an infrared spectrum of this substance determined in theform of a potassium bromide tablets. Absorption maxima (wavelength;cm⁻¹) are as follows.

3470, 3420, 3325, 3200, 2925, 2890, 1655, 1615, 1587, 1565, 1548, 1510,1490, 1455, 1440, 1430, 1388, 1370, 1333, 1317, 1300, 1257, 1230, 1200,1165, 1130, 1100, 1045, 1030, 1010, 975, 955, 910, 860, 810, 795 and743.

(8) Solubility in solvents

This substance is soluble in water, methanol, ethanol and dimethylsulfoxide and hardly or not soluble in other organic solvents such aspropanol, acetone, ethyl acetate, ether and benzene.

(9) Color reactions

Lydon-Smith's reaction and 10% sulfuric acid reaction: positive.

Ninhydrin reaction and Sakaguchi's reaction: negative.

(10) Rf values in thin layer chromatography

This substance shows Rf values of 0.51 and 0.48 in thin layerchromatography with the use of a silica gel thin layer (Kiesel gel 60F254 0.25 nm; mfd. by Merck) and developed with n-butanol/aceticacid/water (4:1:2) and n-butanol/28% aqueous ammonia/water (10:0.5:1),respectively.

(11) ¹ H-nuclear magnetic reasonance spectrum

FIG. 3 shows a ¹ H-nuclear magnetic resonance spectrum determined byusing tetramethylsilane in deuterated dimethyl sulfoxide as an internalreference.

(12) ¹³ C-nuclear magnetic resonance spectrum

Chemical shifts (δ values) of a ¹³ C-nuclear magnetic resonance spectrumdetermined by using dioxane (δ67.4) in deuterated water as an internalreference are as follows:

156.4, 153.5, 149.3, 141.5, 119.4, 82.6, 80.1, 63.3, 59.9 and 45.4.

Based on these data as described above and that obtained by X-raydiffraction, the structure of NK84-0218 has been determined as describedhereinbefore. Up to now, its absolute configuration has not beenclarified.

As will be described hereinafter, the NK84-0218 of the present inventionis expected to be available as pharmaceuticals such as an antibacterialagent, antiviral agent and an antineoplastic agent. It may be formulatedinto pharmaceuticals and administered in conventionally known manners.That is, it may be orally or parenterally (e.g., injection orrectoclysis) administered. It may be formulated into various forms suchas injection, powder, granules, tablet and suppository.

Various pharmaceutically acceptable adjuvants such as carriers,stabilizers, antiseptics, soothing agents and emulsifiers may be used informulating NK84-0218, if desired, so far as they exhibit no adverseeffects.

The content of NK84-0218 in formulation can be varied in a wide range.Generally, a drug comprises 0.01 to 100% by weight, preferably 0.1 to70% by weight, of NK84-0218 and the remainder being adjuvants such as acarrier conventionally used in the art.

The dose of NK84-0218 varies depending on symptoms and other factors.Generally 0.01 to 800 mg of NK84-0218 is administered to an adultpatient in a day. It is preferable to decrease the daily dose when thesubstance must be repetitively administered.

NK84-0218 is usually formulated in the form of a pharmaceuticallyacceptable salt such as hydrochloride of sulfate.

Now biological activities of the NK84-0218 of the present invention willbe described.

1. Antibacterial spectrum

Table 2 shows an antibacterial spectrum of NK84-0218 determined by agarplate dilution method with the use of 0.5% peptone agar.

As shown in Table 2, NK84-0218 exhibits intense effects of inhibitingthe growth of gram-positive bacteria such as Staphylococcus aureus FDA209P, those belonging to the genus Bacillus including B. subtilis PCI219 and B. cereus IAM 1072 and Micrococcus flavus ATCC 10240 but noeffect on gram-negative bacteria.

                  TABLE 2                                                         ______________________________________                                                              Minimum inhibitory                                      Tested bacterium      concentration (μg/ml)                                ______________________________________                                        Staphylococcus aureus EDA 209P                                                                      <0.1                                                    Bacillus subtilis PCI 219                                                                           <0.1                                                    Bacillus subtilis ATCC 6633                                                                         <0.1                                                    Bacillus cereus IAM 1072                                                                            <0.1                                                    Bacillus polymyxa IAM 1210                                                                          <0.1                                                    Bacillus megaterium ATCC 14945                                                                      1.56                                                    Bacillus licheniformis IFO 12107                                                                    6.25                                                    Micrococcus flavus ATCC 10240                                                                       6.25                                                    Micrococcus luteus ATCC 9341                                                                        3.12                                                    Escherichia coli NIHJ >100                                                    Escherichia coli K-12 >100                                                    Klebsiella pneumoniae PCI 602                                                                       >100                                                    Proteus morganii IFO 3168                                                                           >100                                                    Proteus vulgaris IFO 3045                                                                           >100                                                    Proteus mirabilis IFO 3849                                                                          >100                                                    Pseudomonas aernginosa IFO 3445                                                                     >100                                                    Salmonella typhi 901 (MS-1)                                                                         >100                                                    Salmonella paratyphi 1015 (MS-1)                                                                    >100                                                    Salmonella enteritidis G14 (MS-1)                                                                   >100                                                    Enterobacter aerogenes ATCC 13048                                                                   >100                                                    (MS-1)                                                                        Serratia marcescens GM 6484                                                                         >100                                                    Candida albicans NIH 3147                                                                           >100                                                    Mycobacterium smegmatis ATCC 607                                                                    >100                                                    ______________________________________                                    

2. Anti-HeLa cell activity

Table 3 shows the result of an examination on anti-HeLa cell activity ofNK84-0218.

                  TABLE 3                                                         ______________________________________                                                                 Inhibition                                           Concentration                                                                             Cells/plate* ratio    IC.sub.50                                   (μg/ml)  (× 10.sup.5)                                                                         (%)      (μg/ml)                                  ______________________________________                                        100          2.78        93.38                                                25          13.47        10.21    ca. 47.0                                    6.25        13.91        6.94                                                 1.56        14.64        1.26                                                 0.39        14.35        3.58                                                 ______________________________________                                         *The number of cells 72 hours after the addition of NK840218.            

As clearly shown in Table 3, the anti-HeLa cell activity (IC₅₀) of theNK84-0218 of the present invention is approximately 47.0 μg/ml.

(3) Antiviral activity

A medium containing a definite amount of NK84-0218 and herpes simplexvirus-I (HSV-I) 5-10 TC ID₅₀ are added onto a single layer of vero cellsin a microplate having 96 wells and cultured in a 5% by volume carbondioxide incubator at 37° C. for 96 to 120 hours. Then the antiviralactivity of the test substance is determined by microscopicallyobserving the cytopathic effect (CPE) of the HSV-I on the Vero cells.

Table 4 shows the result.

                  TABLE 4                                                         ______________________________________                                        Concentration (μg/well)                                                                     Inhibition ratio (%)                                         ______________________________________                                        10               100                                                          5                78                                                           2.5              22                                                           1.25             11                                                           0.62             0                                                            0                0                                                            ______________________________________                                    

The inhibition is taken as effective when no CPE is observed in a well.For example, when CPE is observed in one well among ten at the sameconcentration, the inhibition ratio is 90%.

As shown in Table 4, the NK84-0218 of the present invention exhibits anantiviral activity.

The acute toxicity (LD₅₀) of NK84-0218 on mice is 100 mg/kg (i.v.) orabove which is lower than those of various known nucleoside antibiotics.

These results as described above indicate that the NK84-0218 of thepresent invention may be expected as a novel pharmaceutical such as anovel antiviral agent, a novel antibacterial agent and a novelantineoplastic agent of a novel mechanism since it exerts an antiviralactivity, an antineoplastic activity and an antibacterial activity ongram-positive bacteria such as Streptococcus aureus and Bacillussubtilis and is significantly different from conventional nucleosideantibiotics in its chemical structure.

To further illustrate the present invention, and not by way oflimitation, the following Examples will be given.

EXAMPLE 1

100 ml of a seed medium (pH 7.2) comprising 2% of soluble starch, 0.5%of glucose, 0.5% of peptone, 0.5% of yeast extract, 0.05% of dipotassiumphosphate, 0.05% of magnesium sulfate and 0.5% of soybean powder waspipetted into a Sakaguchi's reciprocating shake flask of 500 ml involume and sterilized in an autoclave at 120° C. for 20 min. A platinumloopful of NK84-0218 strain (FERM BP-919) slant culture was inoculatedthereto and cultured at 28° C. under shaking at 130 rpm for one day.Separately, 100 ml of a production medium (pH 7.4) comprising 2% ofgalactose, 2% of dextrin, 1.0% of Bacto-Soytone (mfd. by Difco Co.,Ltd.), 0.5% of corn steep liquor (mfd. by Ajinomoto Co., Inc.), 0.2% ofammonium sulfate and 0.2% of calcium carbonate was pipetted into arotary shake Erlenmeyer flask of 500 ml in volume and sterilized in anautoclave at 120° C. for 20 min. 2 ml of the above culturing broth wastransplanted thereto and cultured therein at 27° C. under shaking at 192rpm for four days. The culturing broth was filtered at pH 6.9 to give 18L of a filtrate. (The titer was determined by peptone agar plate cupmethod with the use of Bacillus subtilis PCI 219 as a test organism andthe culturing filtrate was referred to as 1U/ml). The filtrate waspassed through a column of active carbon for chromatography (Mfd. byWako Chemicals Co., Ltd.) to adsorb NK84-0218 thereby. After washingwith water, it was subjected to acetone linear concentration gradientelution with 2400 ml portions of water and 50% aqueous acetone. Activefractions were combined, concentrated in vacuo and lyophilized to give4.72 g (0.6U/mg) of a crude dark brown powder. Then this powder wastreated with methanol and insoluble matters were removed. The residualsoluble portion was dried in vacuo to give 1.91 g (2.0U/mg) of crudebrown powder. This powder was dissolved in 47 ml of water and adsorbedby a column of 150 ml of active carbon. After washing with water, it wassubjected to acetone linear concentration gradient elution with 300 mlportions of water and 50% aqueous acetone. Active fractions werecombined, concentrated in vacuo and lyophilized to give 657 mg (3.9U/mg)of a pale brown powder. Then this powder was dissolved in 2.8 ml ofwater, packed into a column of 600 ml of Avicel® previously equilibratedwith n-propanol/water (97.5:2.5) and eluted with 1200 ml ofn-propanol/water (97.5:2.5), 1200 ml of n-propanol/water (95:5) and 1200ml of n-propanol/water (90:10), successively. Active fractions werecombined, concentrated in vacuo and lyophilized to give 55.5 mg(34.8U/mg) of a pale yellow powder. Then this powder was dissolved in avery small amount of water, packed into a column of 460 ml of SPSephadex® C-25 (Na⁺) previously equilibrated with 0.02M sodium chlorideand eluted with the above sodium chloride. Active fractions werecombined and desalted on active carbon to give 11.2 g (135.3U/mg) of acolorless powder. This powder was dissolved in 1.1 ml of water andallowed to stand overnight at room temperature to give 10.1 mg ofNK84-0218 in the form of colorless needles.

EXAMPLE 2

100 ml of a seed medium (pH 7.2) comprising 2% of soluble starch, 0.5%of glucose, 0.5% of soybean powder (Prorich), 0.5% of peptone, 0.5% ofyeast extract, 0.05% of dipotassium phosphate, 0.05% of magnesiumsulfate and 0.2% of calcium carbonate was pipetted into a rotary shakeErlenmeyer flask of 500 ml in volume and sterilized in an autoclave at120° C. for 20 min. A platinum loopful of Bacillus megaterium NK84-0218(FERM BP-919) slant culture was inoculated thereto and cultured at 27°C. and 200 rpm for 18 hours. Separately, 800 ml of the above medium waspipetted into a rotary shake Erlenmeyer flask of 5000 ml in volume andsterilized in an autoclave at 120° C. for 20 min. 10 ml of the aboveculturing broth was transplanted thereto and cultured therein under thesame condition as described above for additional 18 hours. Furtherseparately, 140 L of a production medium (pH 6.0) comprising 2.0% ofsoluble starch, 1.5% of soybean powder (Prorich; mfd. by Ajinomoto Co.,Inc.), 0.3% of potassumprimary phosphate, 0.2% of sodium secondaryphosphate, 0.0002% of cobalt chloride, 0.0002% of ferrous sulfate and0.05% of magnesium sulfate was introduced into a stainless tank of 200 Lin volume and subjected to pressure steam sterilization at 120° C. for30 min. 2.4 L of the above culturing broth was transplanted theretounder a sterile condition and cultured therein at 37° C. under aeratingat 2/3 VVM and agitating at 270 rpm for 43 hours. After the completionof the culture, the culturing broth was heated to 80° C. for five min,allowed to cool to room temperature and adjusted to a pH value of 3.8with 10% sulfuric acid. Then 16 kg of a filter aid (Dicalite; mfd. byDicalite Orient Co., Ltd.) was added thereto and cells were filteredoff. 290 L of the filtrate (16.4 μg/ml) thus obtained was passed througha column of Dowex 50W×8® (H⁺, 19 L), washed with water and eluted with200 L of 0.5N NH₄ OH/water. Active fractions containing NK84-0218 wereadjusted to a pH value of 9.5 with 4N HCl/water, passed through a columnof Diaion HP-20® (10 L), washed with water and eluted with 30 L of 40%aqueous methanol. Active fractions were combined, concentrated in vacuoand lyophilized to give 18.8 g (199 μg/ml) of crude NK84-0218 in theform of a dark brown powder. Then 380 ml of methanol was added to thispowder and the mixture was stirred at room temperature for one to twohours. Then the methanol was removed in vacuo. To the residual portionsoluble in methanol, water was added to prepare a 2% aqueous solution,which was subsequently passed through a column of Amberlite IRC-50® (H⁺,500 ml), washed with water and eluted with 0.5N aqueous NH₄ OH. Activefractions were concentrated in vacuo and allowed to stand in an iceboxovernight to give 3.16 g (950 μg/ml) of NK84-0218 in the form of crudecrystals. To this crude crystals, 130 ml of water was added and themixture was heated to 40° to 45° C. to thereby dissolve the crystals.Then it was allowed to stand overnight in an icebox to give 2.85 g ofNK84-0218 in the form of colorless needles. 1 mg of crystallineNK84-0218 was referred to as 1000 μg.

FORMULATION EXAMPLE 1

Distilled water was added to 30 parts by weight of the compound (I) togive a total amount of 2000 parts. After dissolving the compound, thesolution was sterilized by filtering through a millipore filter of GStype. 2 g of the filtrate was introduced into a 10 ml vial andlyophilized therein to give a lyophilized injection containing 30 mg pervial of the hydrochloride of the compound (I).

FORMULATION EXAMPLE 2

50 parts by weight of the compound (I), 600 parts of lactose and 330parts of crystalline cellulose were thoroughly mixed and the mixture wascompacted with a Roller Compactor®, ground and sifted to give granulespassing through 16-to 60-mesh sieves.

FORMULATION EXAMPLE 3

30 parts by weight of the compound (I), 120 parts of crystallinelactose, 147 parts of crystalline cellulose and three parts of magnesiumstearate were tabuleted in a V-type mixer to give tablets each weighing300 mg.

What is claimed is:
 1. A process for production of a novel antibioticNK84-0218, which comprises culturing a microorganism, Bacillusmegaterium NK84-0218 strain, in a medium to produce and accumulate theantibiotic NK84-0218 in the medium and collecting the antibioticNK84-0218 therefrom.